This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Phosphorylation of the Kir3 channel by cAMP-dependent protein kinase (PKA) potentiates activity and strengthens channel-PIP2 interactions whereas phosphorylation by protein kinace C (PKC) leads to opposite effects. We utilized mass spectrometry to identify the phosphorylation sites within the Kir3.1 channel subunit upon treatment with protein kinases. We focused on the Kir3.1 C-terminal cytosolic domain that has been reported to be regulated by several modulators. In vitro phosphorylation by PKA exhibited a convincing signal upon treatment with a phosphoprotein stain. The phosphorylated C terminus was subjected to mass spectrometric analysis using MALDI-TOF/MS. Peptide peaks with a mass shift of 80u, which may relate to the addition of a phosphate group, were then subjected to tandem MS (MS2 and MS3) in order to determine the location of the modification. Using this approach, we identified S385 as an in vitro phosphorylation site. Mutation of this residue to an alanyl residue resulted in a reduced sensitivity of Kir3.1* currents to H89 and forskolin, suggesting an in vivo role for this novel site of the Kir3.1 channel subunit in its regulation by PKA. [Note] The following manuscript has been accepted for publication in the European Journal of Physiology: RUSINOVA, R.;SHEN, Y.-M. A.;DOLIOS, G.;PADOVAN, J. C.;YANG, H.;KIRCHBERGER, M.;WANG, R.;LOGOTHETIS, D. E. Mass spectrometric analysis reveals a functionally important PKA phosphorylation site in a Kir3 channel subunit [2008].